The present invention relates to a therapy interfering with cell division, in particular tumor therapy.
The septins are an evolutionary conserved family of proteins required for cytokinesis (reviewed in Cooper and Kiehart, 1996); Longtine et al., 1996; Field and Kellogg 1999). The septins were first described in S. cerevisiae, where mutants in any of the genes CDC3, 10, 11 and 12 are unable to complete cytokinesis, giving rise to multinucleate cells (Hartwell, 1971). Sequence analysis of these four genes by Pringle and co-workers (GenBank accession numbers L16548-L16551) revealed that they encode proteins with similar primary structure, defining the septin family. The known septins range in size from 30 to 60 kDa and contain sequences characteristic of the GTPase superfamily of proteins. In yeast, the septins localize at the site of bud emergence, and indirect but compelling evidence indicates that the septins are components of the neck filaments, a structure previously described by electron microscopy as an ordered array of filaments in close association with the membrane of the bud (Byers and Goetsch, 1976). Neck filaments have only been observed in yeast cells so far. However, and in spite of the differences in the mechanism of cytokinesis between yeast and animal cells, septins were later found to be widespread in higher eukaryotes. A Drosophila septin mutant, pnut, is defective in cytokinesis, and the Peanut protein localizes to the cleavage furrow of dividing cells (Neufeld and Rubin, 1994). Septins with similar localization patterns have also been described in amphibia and mammals (Kinoshita et al., 1997; Xie et al., 1999), and inactivation of septin function by antibody microinjection in cultured mammalian cells and in Xenopus embryos results in cytokinesis defects (Kinoshita et al., 1997; Xie et al., 1999)). Thus, it appears that the septins are involved in an aspect of cell division that has been conserved from yeast to animal cells. Several yeast proteins required for cytokinesis and bud site selection are recruited to the cell division site in a septin-dependent manner (Chant, et al., 1995; Sanders and Herskowitz 1996; DeMarini et al., 1997; Bi et al., 1998), suggesting that the septins can work as a scaffold that directs the correct localization of other proteins. However, the molecular mechanism of septin function in animal cells is still unclear.
Biochemical experiments have revealed that the septins exist as an heteromultimeric complex containing three (in Drosophila), four (in yeast) or more (in mammals) different septin polypeptides (Field et al., 1996; Frazier et al., 1998; Hsu et al., 1998). Moreover, septin complexes can be purified in a filamentous state (Field et al., 1996; Frazier et al., 1998; Hsu et al., 1998). Thus, it is likely that the septins can form filaments in vivo, even if septin-containing filamentous structures (like the neck filaments in yeast) have not yet been described in animal cells. The mechanism and regulation of septin filament assembly remain nevertheless mysterious, as well as many of the physico-chemical properties of the filaments. For instance, it is not known whether the coiled coil domain is involved in polymerization, or whether septin filaments are polar structures. Several septin proteins have been shown to bind, or bind and hydrolyse, guanine nucleotide (Kinoshita et al., 1997; Field et al., 1996; Beites et al., 1999), and mutations that inhibit nucleotide binding also affect septin localization in mammalian cells (Kinoshita et al., 1997; Field et al., 1996). Inter alia, it was shown that the GTP-binding activity of the mammalian septin Nedd5 is necessary for its normal localization (Kinoshita et al., 1997).
However, the precise role of GTP (guanosine triphosphate) binding and hydrolysis in filament formation has not been elucidated.
It was an object of the invention to elucidate the mechanisms involved in septin filament formation in order to provide a novel approach for therapy, in particular cancer therapy, that is based on modulating septin filament formation and thus interfering with cytokines is.
To solve the problem underlying the present invention, septin filament assembly was reconstituted in vitro using a recombinant septin. It was shown that a septin protein can assemble into filaments in a nucleotide-dependent fashion, and that these polymers, like actin filaments and microtubules, are polar structures that assemble with a nucleation mechanism.
The results obtained in the experiments of the present invention show that GTP binding and hydrolysis regulate the filament assembly of a septin protein, in addition, they present a kinetic analysis of septin polymerization.
Thus, the present invention provides the first direct evidence of nucleotide-dependent filament assembly of a septin protein. Based on these observations, it may also be assumed that XSepA filaments assemble with a nucleation mechanism, and that filament growth and stability is regulated by the state of bound guanine nucleotide. In addition, due to the similarity of the septin, both on the sequence level and in terms of their function, it may be assumed that an essentially identical mechanism operates at the level of heteromultimeric septin filaments.
Kinetic analysis of the polymerization reaction has revealed the existence of a lag phase in septin filament assembly FIG. 4A. This feature is indicative of nucleated polymerization, whereby initiation of filaments is energetically unfavored, but under sufficiently high monomer concentrations, nuclei can form. Addition of monomers to these nuclei to yield long filaments subsequently takes place with a higher association constant than the one required for filament initiation (Cantor and Schimmel; 1980)). Actin and tubulin follow such a polymerization mechanism. The existence of a critical concentration (the concentration below which polymerization cannot occur) is a consequence of the kinetic barrier to nucleation. As shown in FIG. 4B, a critical concentration of approximately 0.5 mg/ml, or xcx9c12 xcexcM (in comparison with xcx9c0.2 xcexcM for actin (Mitchison, 1992), or 14 xcexcM for pure tubulin in glycerol buffer (Mitchison and Kirschner, 1984) for XSepA polymerization exists. These data indicate that XSepA filaments are nucleated polymers. On the other hand, a mechanism of linear polymerization (where monomers associate end-to-end with an affinity constant independent of polymer length) has been proposed for septin filaments, based on the length distribution of immunopurified septin complexes (Field et al., 1996); Frazier et al., 1998). It is very likely, however, that in the absence of polymer-stabilising conditions long filaments would not have survived the purification procedure. This could be due to simple mechanical breakage and/or to depolymerization caused by either depletion of nucleotide, or to effects of dilution of the septin complex below the putative critical concentration. In fact, only short filaments (up to 350 nm) were observed in these cases (Field et al., 1996; Frazier et al. 1998) rendering ambiguous a kinetic interpretation of length distribution data.
Two additional properties of actin filaments and microtubules seem to be shared by XSepA filaments. The polymerization dynamics of XSepA in the presence of the slowly hydrolysable GTP analogue, GTP-xcex3-S suggest a role of nucleotide hydrolysis in the destabilisation of the filament structure (FIGS. 4A-4C). Likewise, a number of experimental approaches has established that NTP hydrolysis is linked to destabilisation of the microtubule and actin filament lattices (Mitchison, 1992). Another important feature of these cytoskeletal elements is filament polarity, since it plays a key role in the organization of higher order structures and in the directional transport of molecules along filamentous tracks (Mitchison, 1992); Kirschner and Mitchison, 1986). Using fluorescence microscopy to visualise elongation of pre-assembled septin filaments, it could be observed that the two ends grow with different kinetics (FIGS. 5A and 5B). Although these data provide a strong indication of polarity in septin filaments, proper determination of the growth rates at the different ends could not be achieved. Further analysis of the elongation process is still needed, together with high-resolution structural studies of the septin filaments.
It is interesting to compare the structural features of in vitro-assembled XSepA filaments with those of septin filaments purified from yeast or animal cells. The diameter of septin filaments in these latter cases has been estimated in 7-9 nm (Field et al., 1996; Frazier, et al., 1998; Hsu et al., 1998). Filaments assembled from recombinant XSepA appeared as paired structures formed by what can be interpreted as two filaments of 8-9 nm in width each FIGS. 2A and 2B. If this interpretation is confirmed, the diameter of individual XSepA filaments would be consistent with that of septin filaments purified from cells. Interestingly, paired filaments of length xe2x89xa71500 nm are obtained when the purified yeast septin complex is briefly dialysed from 1M KC1 into a buffer of lower ionic strength. In this latter case, filaments within a doublet were spaced by a gap of 2-20 nm without sign of a bridging structure (Frazier et al. 1998). It is therefore possible that pairing is a conserved feature of septin filaments under conditions that favour elongation. Lateral association, or bundling of septin filaments has also been described in the yeast and Drosophila complexes (Field et al., 1996; Frazier et al., 1998). Similar structures in XSepA filaments FIGS. 2A and 2B have been observed. A higher degree of bundling was evident in filaments assembled from a modified version of XSepA lacking the coiled-coil domain FIGS. 2A and 2B. Thus, it is possible that although not essential for filament formation, the coiled coil domain plays a role in side-to-side filament association.
In Xenopus cell extracts, XSepA is tightly associated with other septins. In this respect, the Xenopus septin complex is similar to septin complexes already described in yeast, insects and mammals (Field et al., 1996; Frazier, et al., 1998; Hsu et al., 1998). On the other hand, since a single septin can efficiently polymerize in vitro a certain degree of redundancy between septin proteins may be expected. It is very likely, particularly in view of the high sequence homology between septins that some, if not all, of the properties described here for XSepA filaments (nucleotide-dependent assembly and disassembly, nucleation mechanism, and polarity) may be shared by the endogenous, heteromultimeric filaments. It is not known whether overexpression of a particular septin could replace for the lack of another septin polypeptide. In yeast, conditional mutations in any of the four septin genes (CDC3, 10, 11 and 12) lead to defects in cytokinesis and to loss of the remaining septins from the division site (Haarer and Pringle, (1987); Kim, Haarer and Pringle, (1991); Ford and Pringle, (1991)). However, analysis of deletion mutants has revealed that CDC10 and CDC11 are each dispensable for viability, and cells lacking CDC10 properly localize Cdc3p and Bud4p and perform cytokinesis (Frazier et al., 1998). Thus, not all the subunits in the yeast septin complex are required for septin function.
Several recent observations have called into question whether ordered filament arrays are required for septin function in budding yeast. Ultrastructural analysis of xcex94cdc10 and xcex94cdc11 cells failed to detect the presence of neck filaments, although as mentioned above, septin-dependent functions are largely maintained in xcex94cdc10 mutants (Frazier et al., 1998). Moreover, septin complexes purified from these mutant strains are biochemically distinct from the wild-type complex. Whereas the wild-type complex seems to assemble into filamentous structures upon dialysis from 1M KC1, mutant complexes fail to do so. Likewise, the non-essential Gin4p kinase is required for certain aspects of septin organization (Longtine et al., (1998)). In xcex94gin4 cells, although cytokinesis occurs normally, the organization of septins at the bud neck appears severely altered and, must likely, incompatible with the normal arrangement of the neck filaments. These data have led to the proposal that assembly of septins into ordered filament arrays is not required for septin function. It has also been suggested, based solely on the inability of xcex94cdc10 complexes to associate into filaments in vitro, that septin filament assembly may be dispensable for function (Field and Kellogg, 1999; Frazier et al., 1998). The evolutionary conservation of the motifs required for GTPase activity strongly suggests that this activity is required for septin function. As it has been shown, GTP binding and hydrolysis are intimately involved in filament formation. A parsimonious resolution to this paradox is that whereas the neck filament arrays are dispensable, filament assembly is nevertheless still required for septin function.
Since septins are clearly implicated in cytokinesis Hartwell et al., 1971; Neufeld et al., 1994), the results of the present invention, which establish a mechanism for septin filament assembly, provide a basis for identifying compounds that interfere with cytokinesis.
The present invention relates, in a first aspect, to a method for identifying a compound that inhibits cytokinesis, characterized in that the compound""s ability to modulate the formation and/or the stability of septins filaments is determined.
The term xe2x80x9cmodulatingxe2x80x9d denotes either xe2x80x9cpartially or completely inhibiting or decreasingxe2x80x9d or xe2x80x9cenhancingxe2x80x9d.
Based on the finding of the invention that septin filament assembly occurs in a GTP dependent fashion, in a preferred embodiment, the compound is tested for its ability to modulate the binding of GTP to septin monomers.
In principle, any septin that has the ability to form filaments in a GTP-dependent manner, either with identical or different septin molecules, may be used, either by itself or in combination with said other septin molecules.
Such a GTP binding assay is a biochemical assay, which may be carried out according to standard protocols, as described, inter alia, by Self et al., 1995. By way of example, this assay may be carried out as follows: In a first step, the septin monomer is incubated, in the presence or absence of the test compound, with GTP that carries a radioactive label (e.g. the commercially available xcex1-32P-GTP or 3H-GTP) or an otherwise measurable, e.g. a fluorescent label, as described, inter alia, by Hazlett et al., 1993, under conditions and for a period of time sufficient to allow saturation of GTP binding sites. The optimal assay conditions may be determined as follows: In a first step, the septin is selected, e.g. recombinant septin A, and the desired protein concentration is established by a standard assay (e.g. the commercially available BioRad assay). In addition, a suitable buffer is selected, e.g. S-buffer, as described in the Examples. Next, time courses of GTP binding are performed using different GTP concentrations to determine the optimal conditions, i.e. nucleotide concentration and incubation time, to reach saturation. Examples for suitable assay conditions are 5 min at a septinA concentration of 1 xcexcM, or 30 min at a septinA concentration of 20 xcexcM at room temperature in S-buffer.
Once the assay has been established, the test compound""s ability to modulate the GTP binding reaction is determined by measuring the amount of bound GTP and comparing the obtained result with that of the control reaction carried out in the absence of the test compound.
The principle of this type of assay is described in the Examples.
The assay may be performed in the high throughput format by automation of the reaction steps, including, in the case of using a radioactive assay, the separation of septin-bound and unbound GTP. In this case, a great number of compounds, e.g. from compound or natural product libraries, are applied to microtiter plates containing the reagents for the binding reaction. After the time required for GTP saturation in the control reaction (absence of test compound), the reaction solution may be filtered through a protein binding matrix, e.g. nitrocellulose, that is arranged in the same geometrical pattern as the original microtiter plate where the reaction took place, and the radioactivity retained in the filters is quantified.
To simplify the assay, the septin(s) may be immobilized on a solid matrix, either via a tag that allows for binding to a suitably modified solid support, e.g. by using a biotinylated septin and a streptavidin-coated microtiter plate. Alternatively, the unmodified septin(s) may be bound to a specially treated plastic or glass surface that allows for unspecific protein binding, e.g. a glass surface treated with 3-aminopropyltriethoxy-silane (Sigma). In this case, the separation of bound versus unbound GTP is achieved by simply washing away the unbound nucleotide and measuring the amount of label remaining on the plate.
Alternatively to GTP binding, the assay may be based on determining the compound""s ability to modulate GTP hydrolysis. In principle, such an assay is carried out in a similar manner as described above for the GTP binding assay, with the difference that the test compound is added to the reaction after GTP saturation is complete (since GTP binding to septins has to occur prior to GTP hydrolysis, the assay design has to ensure that GTP binding occurs). The GTP molecule must be labeled in the gamma-phosphate group (e.g. gamma-32P-GTP) in order to specifically detect hydrolysis. The readout, which may be done in the same way as described above for the GTP binding assay, may be, preferably, the amount of non-hydrolized GTP, or alternatively, the amount of released orthophosphate. The upscale of the assay to the high throughput format can be done according to the same principles as described above.
In the above-described assays that measure GTP binding to septins or GTP hydrolysis upon binding, the septin molecules employed may be identical or different. Preferably, the assay is carried out by using human septins, using as many different septins (some of them to be identified yet, e.g. by the Human Genome Project) as possible, either alone or in combination, in order to mimic the physiological situation as closely as possible. However, as long as not all human septins are available, the assay may conducted with septins from other organisms, preferably from vertebrates, e.g. Xenopus laevis.
Alternatively to using the full-length protein, a truncated version may be used, as long the GTPase activity is maintained, e.g. a fragment lacking the C-terminal sequence which, in the case of XseptinA has been shown to be dispensible for filament formation.
A number septins that are suitable for use in a GTP binding or hydrolysis assay are available, they have been cloned from various species. Preferably, the septins are employed as recombinant proteins obtained by expression in suitable hosts, e.g. in E. coli or in insect cells, according to conventional methods.
Examples for septins that may be employed are hNEDD5 (GenBank accession number D63878), PNUTL2 (GenBank accession number NMxe2x80x94004574) and CDCREL-1 (GenBank accession number AF006998 U07983).
Alternatively to determining a compound""s effect on GTP binding or GTP hydrolysis, it may be determined whether the compound promotes or inhibits septin filament formation, or enhances or reduces the stability of septin filament.
A suitable assay comprises incubating septin molecules, which may be identical or different, and monitoring the formation of filaments, e.g. by fluorescence measurement. In such an assay, the septin monomers carry a fluorescent label whose fluorescence increases when the agent is arranged in a precise order, as it occurs e.g. upon filament assembly. An example for such a fluorescent label is a pyrene, e.g. N-(1-pyrenyl)iodoacetamide, which was described by Kouyama et al., 1981, for conjugating actin to follow polymerization. This method can be applied in the present invention to monitor septin filament formation.
A compound""s modulating effect on polymerization is determined by measuring its effect on the increase of the fluorescence that is observed in a control experiment carried out in the absence of the compound.
An assay of this type can be established as follows:
The fluorescence-labeled septin molecules, either identical or different, are incubated, in the presence or absence (control sample) of the test compound, with GTP under conditions that allow polymerization in the control sample (in the case of septinA, the minimal conditions are 0.6 mg/ml septin and 0.1 mM GTP in S-buffer for 5 min) and are sufficient for a measurable increase in the fluorescent signal. The readout of the assay; which shows a test compound""s effect on filament formation, is the difference in fluorescence between the test sample and the control sample.
In an alternative assay, the test compound""s effect on septin filament stability can be determined by a similar method, with the difference that the test compound is added to reaction mixture after filament formation has been completed. Upon further incubation with the test compound the fluorescence will either decrease (in the case of the compound""s filament destabilizing effect) or increase (in the case of the compound""s filament stabilizing effect).
The above-described assays, which determine whether the compound promotes or inhibits septin filament formation, or whether it enhances or reduces the stability of septin filament, can be employed as a primary screen or, alternatively, as a secondary screen following a primary screen based on GTP binding and/or GTP hydrolysis. Such a secondary screen may be followed by a detailed characterization of the compound""s effect on filament formation/stability, as assayed by standard sedimentation, fluorescence or electron microscopy procedures. The secondary assay and the subsequent filament characterization will confirm that a test compound does not only effect GTP binding/hydrolysis, but also filament formation/stability, which appears to be the septins"" fundamental biological role in cytokinesis and is therefore the mechanism relevant for therapeutical intervention.
Compounds that have been identified in the above-defined screens due to their ability to modulate the nucleotide binding or hydrolysis and/or polymerization of septin monomers in vitro are candidates for drugs that affect the function of septins in vivo.
Since septins have an essential role in cell division, modulators of septin filament assembly/stability have the potential to perturb cytokinesis. In tumor cells, this effect may result in a decrease or a stop of tumor growth. In addition, inhibition of cytokinesis may cause the activation of a cell cycle arrest check point that will trigger apoptosis of the tumor cells.
By way of example, the candidate compounds can be assayed for their effect on cytokinesis and other cellular processes in tissue culture of normal or transformed cells. To test the inhibition of tumor cell proliferation, primary human tumor cells are incubated with the compound identified in the screen and the inhibition of tumor cell proliferation is tested by conventional methods, e.g. bromo-desoxy-uridine or 3H incorporation.
Compounds that exhibit an anti-proliferative effect in these assays may be further tested in tumor animal models and used for the therapy of tumors.
Thus, in a further aspect, the invention relates to compounds identified in the above screens for the therapy of tumors.
In addition to their role in cytokinesis, septins have been shown to be highly expressed in brain, where they are associated with molecules of the secretory pathway. Thus, the septins appear to be involved in neurosecretion events (Hsu et al., 1998; Beites et al., 1999). Modulators of septin filament assembly/stability may therefore be useful for the treatment of neurodegenerative disorders, e.g. Alzheimer""s disease or Huntington disease, and stroke.
Thus, in a further aspect, the invention relates to compounds identified in the above screens for the therapy of neurodegenerative diseases.
Toxicity and therapeutic efficacy of the compounds identified as drug candidates by the method of the invention can be determined by standard pharmaceutical procedures, which include conducting cell culture and animal experiments to determine the IC50, LD50, the ED50. The data obtained are used for determining the human dose range, which will also depend on the dosage form (tablets, capsules, aerosol sprays, ampules, etc.) and the administration route (oral, buccal, nasal, paterental or rectal). A pharmaceutical composition containing the compound as the active ingredient can be formulated in conventional manner using or more physiologically active carriers and excipients. Methods for making such formulations can be found in manuals, e.g. xe2x80x9cRemington Pharmaceutical Sciencesxe2x80x9d.